The present invention relates to a novel growth factor from bovine milk, milk products or milk product extracts. The present invention also relates to the use of recombinant DNA technology to isolate, clone and sequence nucleic acids encoding the mature and precursor forms of the growth factor and, in addition, to the use of these nucleic acids in the recombinant production of the growth factor.
Growth factors are implicated in a wide range of physiological and pathological processes such as cell communication, growth and development, apoptosis, embryogenesis, initiation of the immune response, cell survival and differentiation, wound healing, and cancer. Justifiably, there is a great deal of interest in isolating, characterising and defining the functional mechanisms of growth factors, not only in understanding the basic mechanisms behind normal growth control, but also because of their potential therapeutic use.
Growth factors may also comprise an essential component of defined media used in the growth of cells in culture by the biotechnology industry. For many years animal sera have been used to supplement culture media to provide essential components for cell growth. Foetal bovine serum (FBS) is most commonly used, however there are market and regulatory concerns about the safety of animal and human sourced proteins in pharmaceutical manufacturing processes.
Despite the considerable progress made using milk based alternatives to serum, the safety concerns surrounding mammalian body fluids as supplements for cell culture media (as a consequence of the potential for infection with latent pathogens (eg. bovine spongiform encephalopathy (BSE)) means that these are considered unsafe. Therefore attention has focussed on the development of completely defined cell culture media containing growth factors of known origin and defined purity. This means that the growth factor component of cell culture media is provided by purified native or recombinant molecules.
Milk is an important nutrient required for the growth and development of an infant. It is a source of nutrients such as casein, lactoferrin, lactalbumin, lactose and various other compounds such as vitamins, ions, enzymes etc. A number of growth factors have been identified in human and bovine milk. These include Insulin-like growth factor I and II, fibroblast growth factors, transforming growth factor xcex2, and platelet-derived growth factor. Although epidermal growth factor is present in human milk it has not been convincingly demonstrated in bovine milk (lacopetta et al, Acta Paediatr, 81, 287, (1992)). Several roles have been proposed for milk-derived growth factors including development and differentiation of the mammary gland (Collier et al, Livestock Production Science, 35, 21, (1993)), regulation of the developing neonatal immune system (Ishizaka et al. Cellular Immunol, 159, 77, (1994)), gastrointestinal growth and maturation (Read et al, Pediatric Research, 18, 133, (1984)), and possible actions in other organs. Milk-derived growth factors have also been shown to support the growth of a variety of cells in culture. In addition, bovine milk whey, the by-product of cheese manufacture, can also support the growth of mammalian cells in the short or long term (Derouiche et al, Lait, 70, 313, (1990); Damerdji et al, Biotechnology Tech, 2, 253, (1988)) and has been shown to possess antitumor activity (Bounous et al, Clin. Invest. Med. 11, 312, (1988)). The prior art also includes Australian patent 645589 to the present applicant which describes the use of a bovine milk whey extract, containing a plurality of cell growth stimulating factors, as a replacement for serum in mammalian cell culture.
It is an object of the present invention to overcome, or at least alleviate, one or more of the difficulties or deficiencies associated with the prior art. Accordingly, in a first aspect of the present invention there is provided a novel growth factor, hereinafter referred to as Bovine Milk Growth Factor (BMGF) obtainable from bovine milk, milk products or milk product extracts. The term xe2x80x9cmilkxe2x80x9d as used herein refers to lactational secretions of human or animal origin.
The BMGF may be in a substantially pure or partially pure form. The term xe2x80x9csubstantially purexe2x80x9d as used herein to describe the purity of BMGF means at least 70% pure.
The term xe2x80x9cpartially purexe2x80x9d as used herein to describe the purity of BMGF means a specific activity greater than 0.3 units per milligram protein.
The term xe2x80x9cone unitxe2x80x9d as used herein as a measure of BMGF activity is defined as the amount of BMGF required to compete for 50% of the binding of 125I-labelled recombinant human epidermal growth factor to AG2804 cells under the assay conditions described in Example 1 of this specification.
The native BMGF in its glycosylated form may have a molecular weight of approximately 21 to 25 kDa as determined by SDS-PAGE. It has the ability to stimulate the proliferation of fibroblasts and/or epithelial cells.
In a preferred form of this aspect of the invention the BMGF has an amino acid sequence as follows:
DGNSTRSPEDDGLLCGDHAENCPATTTQPKRRGHFSRCPKQYKHYCIKGRCRFVVAEQTPSCVCDEGYAGARCERVDLFY (SEQ ID NO:2)
The BMGF disclosed herein may be a member of the epidermal growth factor family of growth factors. It contains eighty amino acid residues and eight half cysteines, a pattern that has also been described for an epidermal growth factor-like molecule purified from the conditioned media of mouse pancreatic beta tumor cells (Shing et al, Science, 259, 1604, (1993)). The amino acid sequence of the human form of this factor, termed, betacellulin, has also been deduced from a nucleotide sequence obtained from a human adenocarcinoma cell line (Sasada et al, Biochem. Biophys. Res. Commun. 190, 1173, (1993)). It is likely, based on the sequence homologies between BMGF and mouse betacellulin (58 identical residues) and with human betacellulin (72 identical residues), that BMGF is the bovine form of the betacellulin. Clearly, it is not the same molecule. The BMGF isolated from bovine cheese whey extract has a molecular mass of 21-25 kDa, which is substantially smaller in size than the 32 kDa reported for the natural mouse betacellulin.
The present invention also includes within its scope precursor forms of BMGF and functionally active fragments (peptides) of BMGF. Such fragments may have enhanced or diminished growth stimulatory activity and/or may expand or limit the range of cells responsive to BMGF""s growth stimulatory activity. They may find useful applications in areas such as, but not limited to, the repair or prevention of gut damage or in wound healing.
They may be produced by methods known to those skilled in the art. Procedures at the genetic level such as (but not limited to) site-directed mutagenesis or at the protein level such as (but not limited to) chemical modification are within the scope of the invention.
In a preferred form of this aspect of the invention the fragments of BMGF may include the following amino acid sequences:
DGNSTRSPEDDGLLCGDHAENCPATTTQPKRRGHF (SEQ ID NO:3); or
GYAGARCERVDLFY (SEQ ID NO:4); or
DGNSTRSPEDDGLLCGDHAENCPATTTQPK (SEQ ID NO:5); or
RRGHFSRCPK (SEQ ID NO:6); or
QYK (SEQ ID NO:7); or
HYCIK (SEQ ID NO:8); or
GRCRFVVAEQTPSCVCDEGYAGARCERVDLFY (SEQ ID NO:9)
The present invention also provides BMGF that is substantially non-glycosylated. This may be prepared, for example, by subjecting glycosylated BIZGF to an enzymatic deglycosylation step and recovering the de-glycosylated form. The BMGF in its non-glycosylated or partially non-glycosylated form may have a molecular weight of approximately 9-14 kDa as determined by SDS-PAGE.
In a further preferred form of this aspect of the invention, the BMGF is obtained from bovine milk, bovine milk products or bovine milk product extracts. Moire preferably it is obtained from cheese whey, most preferably from bovine cheese whey extract.
In a further aspect of the present invention, there is provided a process for the isolation, in a substantially pure or partially pure form, of BMGF from bovine milk or milk products. More preferably it is isolated from cheese whey or a cheese whey extract, most preferably from bovine cheese whey or bovine cheese whey extract. The BMGF in its glycosylated form has a molecular weight of approximately 21 to 25 kDa as determined by SDS-PAGE. It may have the ability to stimulate the proliferation of fibroblasts and/or epithelial cells, and/or osteoblast cells.
In a preferred form of this aspect of the invention the BMGF has an amino acid sequence as follows:
DGNSTRSPEDDGLLCGDHAENCPATTTQPKRRGHFSRCPKQYKHYCIKGRCRFVVAEQTPSCVCDEGYAGARCERVDLFY (SEQ ID NO:2)
The process of isolating substantially pure BMGF in this aspect of the invention may include subjecting the milk or a milk product or milk product extract to various purification steps such as ion-exchange chromatography, size-exclusion chromatography, affinity chromatography and reverse-phase high performance liquid chromatography and/or if necessary further purification processes. A xe2x80x9cmilk productxe2x80x9d may include cheese whey, skim milk, acid (Casein) whey and colostrum. Preferably, the milk or milk product is subjected to a process outlined in AU645589 to provide a milk product extract prior to the purification steps outlined above. The contents of AU645589 are incorporated herein. A xe2x80x9cmilk product extractxe2x80x9d is defined herein as an extract prepared from milk or a milk product by a process described in Australian Patent AU645589. By way of clarification, the defining of a milk product extract encompasses a cheese whey extract.
Sample fractions collected from each purification step, and which show biological activity in a radioreceptor assay using AG2804 cells are pooled and forwarded to the next purification step. Sample material from each of the pooled fractions may be subjected to a dose response analysis, thereby allowing for an estimate of the amount of material required to elicit a 50% response of the maximal activity in the said assay. This said amount, in conjunction with the values for the amount of material in each pool, may be used to calculate the yield of purification recovery, and specific activity at each step of the process. Homogeneity of substantially pure BMGF may be demonstrated by:
migration as a single band of approximately 21-25 kDa following SDS-PAGE,
N-terminal sequence analysis,
mass spectroscopy and/or
specific binding to an EGF-receptor
In a preferred form of this aspect of the invention there is provided a method of isolating substantially pure BMGF which method includes
providing bovine milk, milk product or milk product extract;
subjecting the bovine milk, milk product or milk product extract to ultrafiltration to obtain a first fraction;
subjecting the first fraction to anion exchange chromatography to obtain a second fraction;
subjecting the second fraction to gel filtration chromatography to obtain a third fraction;
subjecting the third fraction to reverse phase high performance liquid chromatography ((RP)HPLC) to obtain a fourth fraction;
subjecting the fourth fraction to affinity chromatography to obtain a fifth fraction;
subjecting the fifth fraction to (RP)HPLC to obtain a sixth fraction;
subjecting the sixth fraction to (RP)HPLC to obtain the substantially pure BMGF.
In a further preferred form of this aspect of the invention there is provided a method of isolating substantially pure BMGF which method includes
providing a milk product extract prepared according to AU645589;
subjecting the milk product extract to acidification;
subjecting the acidified milk product extract to ultrafiltration to obtain a first fraction;
subjecting the first fraction to anion exchange chromatography to obtain a second fraction;
subjecting the second fraction to gel filtration chromatography to obtain a third fraction;
subjecting the third fraction to reverse phase high performance liquid chromatography ((RP)HPLC) to obtain a fourth fraction;
subjecting the fourth fraction to affinity chromatography to obtain a fifth fraction;
subjecting the fifth fraction to (RP)HPLC to obtain a sixth fraction;
subjecting the sixth fraction to (RP)HPLC to obtain the substantially pure BMGF.
Preferably, the milk, milk product or milk product extract is subjected to an acidification step. Preferably the acidification is conducted prior to ultrafiltration. More preferably the acidification is conducted at a pH 2.5.
Preferably the ultrafiltration is performed using a membrane with an exclusion limit of approximately 50-150 kDa, more preferably approximately 100 kDa.
Preferably the anion exchange chromatography is performed using an agarose-based anion exchange column.
Preferably the gel filtration is performed using a column which separates proteins having molecular weights in the range approximately 3 kDa to 70 kDa.
Preferably the first: (RP)HPLC is performed using a C4 or C18 matrix.
Preferably the affinity chromatography is performed using a heparin/agarose-based affinity column.
Preferably the second (RP)HPLC is performed using a C4 or C18 matrix.
Preferably the third (RP)HPLC is performed using a C4 or C18 matrix.
As an alternative to producing a milk product extract prepared according to AU645589 as the starting material, a bovine milk or milk product may be utilised. If this approach is adopted a preliminary purification step may be used to remove the fat, solids and acidic proteins before the anion exchange chromatography step.
The substantially purified BMGF may be used in the production of polyclonal and monoclonal antibodies which recognise and/or bind to BMGF and this is considered within the scope of the present invention.
Accordingly, in a further aspect of the present invention there is provided a polyclonal or monoclonal antibody against BMGF.
Various procedures are known in the art which may be used for the production of antibodies to epitopes of BMGF. Various host animals may be used in the production of these BMGF antibodies following immunisation with BMGF protein including but not restricted to rabbits, mice, goats etc. Adjuvants may be used to increase the immunological response, depending on the host species, and may include but are not restricted to Freunds (complete and incomplete). BMGF monoclonal antibodies may be prepared by using techniques which enable the continuous production of antibody molecules by cell lines in vitro. These may include, but are not limited to, the hybridoma technique (Kohler and Milstein, Nature 256, 495, (1975)). Antibodies to BMGF may find use in the detection of mature and precursor forms of BMGF in various tissues, body fluids and cell lines, for example in screening assays for the growth factor, and in the affinity purification of BMGF protein.
In a still further aspect of the present invention there is provided a nucleic acid encoding BMGF or fragments thereof encoding functionally active fragments of BMGF. The nucleic acid may encode mature or precursor forms of BMGF.
Preferably the nucleic acid has the sequence shown in FIG. 5.
Due to the degeneracy of the genetic code, other nucleic acid sequences which encode the same or functionally equivalent amino acid sequence are included within the scope of the current invention. Such alterations of the BMGF nucleotide sequence may include substitutions of different nucleotides resulting in the same or a functionally equivalent gene product. Also included within the scope of this invention are nucleic acid sequences having deletions and/or additions and which result in a functionally equivalent gene product.
In a still further aspect of the present invention, there is provided a method for isolating a nucleic acid encoding BMGF or fragments thereof encoding functionally active fragments of BMGF. The nucleic acid may encode mature or precursor forms of BMGF.
The nucleic acid encoding BMGF may be obtained from cell sources that produce BMGF activity. For example, kidney cells may be used as the source of the nucleic acid. The nucleic acid encoding BMGF is preferably obtained by (but is not restricted to) reverse transcription of BMGF mRNA into complementary cDNA and subsequent Polymerase Chain reaction (PCR) amplification using oligonucleotide primers containing nucleic acid sequences encoding portions, preferably the extreme N and C terminal portions of the mature or precursor protein. Preferably the oligonucleotide primers have the following sequences or substantially homologous sequences:
5xe2x80x2 ATC TAG GTT ACC ATG GAT GGG AAT TCA ACC AGA 3xe2x80x2 (SEQ ID NO:10)
5xe2x80x2 ATC TAG GTT ACC GGC GAT GGG AAT TCA ACC AGA 3xe2x80x2 (SEQ ID NO:11)
5xe2x80x2 CTA GAT AAG CTT TCA TCA GTA AAA CAA GTC AAC TCT 3xe2x80x2 (SEQ ID NO:12)
Preferably the oligonucleotide primers include restriction enzyme sites to facilitate directional cloning of the nucleic acid.
More preferably, the primers include the following nucleotide sequence:
5xe2x80x2 GGG AAT TCA ACC AGA 3xe2x80x2 (SEQ ID NO:13)
5xe2x80x2 GTA AAA CAA GTC AAC TCT 3xe2x80x2 (SEQ ID NO:14).
Most preferably, the primers are:
5xe2x80x2 GGG AAT TCA ACC AGA AGT CCT GAA 3xe2x80x2 (SEQ ID NO:15)
5xe2x80x2 GTA AAA CAA GTC AAC TCT CTC ACA CCT 3xe2x80x2 (SEQ ID NO:16)
Thus, in a preferred form of this aspect of the invention there is provided a method for isolating a nucleic acid encoding BMGF or fragments thereof encoding functionally active fragments of BMGF, said method including
providing a source of cells having BMGF activity;
treating the cells to obtain mRNA therefrom;
treating the mRNA thus obtained to produce cDNA therefrom; and
amplifying the cDNA thus obtained by PCR using oligonucleotide primers to produce the nucleic acid.
Other methods, well known to those in the art, for obtaining nucleic acids encoding proteins exist, and the use of these methods to obtain nucleic acids encoding mature or precursor forms of BMGF is also included within the scope of the current invention. These methods may include (but are not restricted to) either chemically synthesising the nucleic acid from knowledge of the nucleic acid or amino acid sequence of the mature and/or precursor forms of BMGF or screening a cDNA and/or genomic library, preferably a bovine library, with isotopically or non-isotopically labelled nucleic acid sequences homologous to nucleotide sequence encoding part or all of the mature and/or precursor forms of BMGF.
Using standard techniques of recombinant DNA technology, well known to those skilled in the art, the nucleic acid encoding the mature or precursor forms of BMGF may be cloned into an appropriate vector, for example an expression vector.
Accordingly, in a further aspect of the present invention there is provided a vector including a nucleic acid encoding BMGF or fragments thereof encoding functionally active fragments of BMGF.
The nucleic acid may encode mature or precursor forms of BMGF.
Preferably the nucleic acid has the sequence shown in FIG. 5.
A large number of vector-host systems are available and these include (but are not restricted to) plasmids such as pBR322 or pUC derivatives, or bacteriophage such as lambda derivatives.
The vector of the present invention may be used to express recombinant BMGF in host cells.
Accordingly in a still further aspect of the present invention there is provided recombinant BMGF.
The recombinant BMGF may be in a substantially pure form. Preferably it is at least about 70% pure, more preferably at least about 90% pure, most preferably at least about 99% pure. The recombinant BMGF may have a molecular weight of approximately 9 kDa in the nonglycosylated form as determined by SDS-PAGE. It may have the ability to stimulate the proliferation of fibroblasts and/or epithelial cells and/or osteoblast cells.
In a preferred form of this aspect of the invention the BMGF has an amino acid sequence as follows:
DGNSTRSPEDDGLLCGDHAENCPATTTQPKRRGHFSRCPKQYKHYCIKGRCRFVVAEQTPSCVCDEGYAGARCERVDLFY (SEQ ID NO:2)
The present invention also includes within its scope precursor forms of recombinant BMGF and functionally active fragments (peptides) of recombinant BMGF.
In a preferred form of this aspect of the invention the fragments of BMGF may include the following amino acid sequences or substantially homologous sequences:
DGNSTRSPEDDGLLCGDHAENCPATTTQPKRRGHF (SEQ ID NO:3)
GYAGARCERVDLFY (SEQ ID NO:4); or
DGNSTRSPEDDGLLCGDHAENCPATTTQPK (SEQ ID NO:5); or
RRGHFSRCPK (SEQ ID NO:6); or
QYK (SEQ ID NO:7); or
HYCIK (SEQ ID NO:8); or
GRCRFVVAEQTPSCVCDEGYAGARCERVDLFY (SEQ ID NO:9).
The present invention also provides a method for producing recombinant BMGF, said method including
providing a vector including a nucleic acid encoding BMGF or fragments thereof encoding functionally active fragments of BMGF; and
a host cell;
introducing said vector into said host cell;
expressing said recombinant BMGF; and
isolating said recombinant BMGF.
The vector may be introduced into the host cell by methods such as (but not restricted to) transformation, transfection, electroporation and infection. Host cells may include but are not restricted to bacteria such as E.coli cells.
The recombinant BMGF may be expressed as a fusion protein. The fusion proteins formed according to this aspect of the present invention, may be isolated as inclusion bodies within the host cell.
It will be understood that the recombinant BMGF so formed may be isolated, preferably following disruption of the host cell, by conventional methods of polypeptide purification well known to those skilled in the art, utilising techniques such as ion-exchange chromatography, size-exclusion chromatography, affinity chromatography, reverse-phase high performance liquid chromatography and/or if necessary further purification processes. The recombinant BMGF may be isolated as a fusion protein or cleaved from its fusion partner using conventional methods well known to those in the art.
In a preferred form of this aspect of the invention, the recombinant BMGF may be prepared as a fusion protein according to Australian Patent 633099 to the applicant, the entire disclosure of which is incorporated herein by reference, wherein a portion of porcine growth hormone is linked to the N-terminal sequence of BMGF, optionally through a cleavable sequence.
The BMGF of the present invention, including the naturally-derived mature or precursor forms of BMGF, and recombinant BMGF protein isolated as either the mature or precursor form with or without its fusion partner (and mutants, analogues, fragments and derivatives thereof) may be utilised:
in the growth and/or proliferation of mammalian cells and more organised structures such as skin in vitro either alone or in combination with other factors (such as or not restricted to IGF-I or IGF-I analogues) or, for example as a supplement to foetal serum
in the enhancement of wound healing and/or tissue repair, eg. in the treatment of surface wounds, either alone or in combination with other factors
in the prevention, amelioration or treatment of conditions associated with impaired gut barrier function, such as inflammatory bowel disease and mucosal immunity, either alone or in combination with other factors
as a supplement in infant milk formulae
in the prevention, treatment or amelioration of peridontal disease
in cosmetic applications.
Accordingly, the present invention provides a composition for promoting the growth and/or proliferation of mammalian cells and tissues, said composition including an effective amount of BMGF, or mutants, analogues and derivatives of BMGF, precursor and fusion protein forms of BMGF and functionally active fragments (peptides) of BMGF; together with a culture medium, preferably a defined culture medium.
The present invention also provides a method for promoting the growth and/or proliferation of mammalian cells and tissues, said method including growing said cells or tissues in a culture medium including an effective amount of BMGF, or mutants, analogues and derivatives of BMGF, precursor and fusion protein forms of BMGF and functionally active fragments (peptides) of BMGF.
The term xe2x80x9ceffective amountxe2x80x9d as used herein in methods of use for BMGF means an amount sufficient to elicit a statistically significant response at a 95% confidence level (p less than 0.05 that the effect is due to chance alone).
In a further aspect of the present invention there is provided a method for promoting the growth and/or proliferation of mammalian cells and tissues, said method including growing said cells or tissues in a culture medium including an effective amount of BMGF, or mutants, analogues and derivatives of BMGF, precursor and fusion protein forms of BMGF and functionally active fragments (peptides) of BMGF, together with an effective amount of an IGF that produces a synergistic response.
The IGF or insulin-like growth factor that is included in the culture medium of this further aspect of the present invention may be IGF-I, IGF-II or a functionally effective mutant or analogue of IGF such as LR3IGF-1 described in Australian Patent 633099 to the applicant.
In a further aspect of the present invention there is provided a composition for the treatment of surface wounds, said composition including an effective amount of BMGF, or mutants, analogues and derivatives of BMGF, precursor and fusion protein forms of BMGF and functionally active fragments (peptides) of BMGF; together with a pharmaceutically acceptable excipient, diluent or carrier
The present invention also provides a method for enhancing wound healing and/or tissue repair, said method including administering to a patient in need thereof an effective amount of BMGF, or mutants, analogues and derivatives of BMGF, precursor and fusion protein forms of BMGF and functionally active fragments (peptides) of BMGF.
There are no limitations on the type of wound that may be treated, and these may include (but are not restricted to): surface ulcers (eg. pressure, venous stasis, diabetic and atherosclerotic ulcers), burns or accidental wounds (such as lacerations and incisions) and wounds to epithelial lined organs such as the stomach and intestine (large and small) and corneal injury to the eye. The application of BMGF to wound sites may be in the form of (but is not restricted to) a powder, gel, ointment or bandages and other wound dressings incorporating BMGF. The BMGF may be applied to wounds either alone or in a mixture including other growth factors, and may be in combination with other ingredients, such as adjuvants, carriers and solubilising agents. The concentration of BMGF in the composition is not critical but should be enough to induce cell proliferation, particularly epithelial cell proliferation.
The gastrointestinal tract (gut) is constantly challenged by potentially harmful substances present in the intestinal lumen and subsequently, maintenance of a normal mucosal barrier is crucial in both adult and infant life. Barrier function may be compromised by damage to the epithelial surface, for example as occurs during highdose chemotherapy, infection and trauma. In addition, a pro-inflammatory response, generated after sensitisation to normal and luminal antigens, may lead to epithelial damage and increased permeability. This immune dysfunction is thought to play a significant role in the pathogenesis of disorders including coeliac and inflammatory bowel disease. Given the epithelial-cell stimulatory activity of BMGF, a still further application of the present invention is in the treatment, amelioration or prevention of conditions associated with impaired gut function, such as inflammatory bowel disease and mucosal immunity, either alone or in combination with other growth factors, preferably as an enteral formulation.
Accordingly, in a still further aspect of the present invention there is provided a composition for the prevention, amelioration or treatment of conditions associated with impaired gut function, said composition including an effective amount of BMGF, or mutants, analogues and derivatives of BMGF, precursor and fusion protein forms of BMGF and functionally active fragments (peptides) of BMGF; together with a pharmaceutically acceptable excipient, diluent or carrier.
The present invention also provides a method for preventing, ameliorating or treating conditions associated with impaired gut function, said method including administering to a patient in need thereof an effective amount of BMGF, or mutants, analogues and derivatives of BMGF, precursor and fusion protein forms of BMGF and functionally active fragments (peptides) of BMGF.
The present invention also provides a method for preventing or treating periodontal disease said method including administering to a patient in need thereof an effective amount of BMGF, or mutants, analogues and derivatives of BMGF, precursor and fusion protein forms of BMGF and functionally active fragments (peptides) of BMGF.
There is also provided a use of BMGF in a cosmetic application, said use including administering to a patient in need thereof an effective amount of BMGF, or mutants, analogues and derivatives of BMGF, precursor and fusion protein forms of BMGF and functionally active fragments (peptides) of BMGF.
The present invention also provides an infant formula including BMGF, or mutants, analogues and derivatives of BMGF, precursor and fusion protein forms of BMGF and functionally active fragments (peptides) of BMGF; together with nutrient components. Preferably the infant formula is a milk formula.
Throughout the description and claims of this specification, the word xe2x80x9ccomprisexe2x80x9d and variations of the word, such as xe2x80x9ccomprisingxe2x80x9d and xe2x80x9ccomprisesxe2x80x9d, is not intended to exclude other additives, components, integers or steps.
The present invention will now be more fully described with reference to the accompanying Examples and drawings. It should be understood, however, that the description following is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above.